1
I N T RO DU C T IO N
Residue analysis for food safety involves
detection and/or quantification of trace amount of
contaminants that may be present in a food, feed,
or biological matrix (e.g., meat, hair, tea, urine).
The contaminants may include pesticides, veterinary
drugs, growth promoters, and their metabolites. Their
presence in food is a potential threat to consumer
health, and international organizations have issued
regulations on these substances in food products.1
R E SU LT S A N D D IS C U S S IO N
Tandem mass spectrometry (MS/MS) coupled to liquid chromatography (LC)
is currently the most widely used technique for organic residue analysis.
This is mainly because of its highly sensitive and selective multiple reaction
monitoring (MRM) mode of measurement. In this mode, pre-selected ions
(precursor ions) are fragmented and their characteristic fragment ions (product
ions) are detected and quantified. It has high sensitivity, high selectivity, wide
detector dynamic range, and it is very suitable for trace level detection and
quantification. Multiple residues can be analyzed in a single run by setting up
multiple timed MRM windows (or MRM channels). Using this approach, multiple
residues of over 400 substances may be screened with one injection and
confirmed with two injections by LC-MS/MS analysis.2
Although LC-MS/MS is the most used MS technique in residues analysis
laboratories, it has its own limitations. First, it is not suitable for non-targeted
analysis. Prior knowledge of the characteristic transitions (precursor ions
to product ions) for a substance is a prerequisite for MRM measurement. It
is impossible to predict the characteristic MRM transitions of the unknown
beforehand. Secondly, there are a finite number of MRM transitions that can be
monitored by LC-MS/MS technique in a single run, since each transition needs
dedicated mass analyzer machine time.
Quadrupole Time-of-Flight (Q-TOF) mass spectrometry is a hybrid technique
that combines the benefits of the MS/MS technique and the TOF accurate
mass MS technique. It offers an alternative but attractive approach for residue
analysis. Modern Q-TOF MS instruments, such as the Waters® Xevo® QTof mass
spectrometers, provide accurate mass determination, high mass resolving power,
improved dynamic range, and speed. They are capable of analyzing virtually
unlimited numbers of substances simultaneously without any prior knowledge of
the analytes. This not only avoids tedious MRM tuning for multiple compounds
as a tandem quadrupole instrument requires; but more importantly, it opens
the door for non-targeted or unknown analysis. This is especially useful when
analytes have no reference standards available. Furthermore, the full scan mass
spectrometric data obtained by TOF MS can be analyzed retrospectively when
new substances of interest emerge. This kind of “collect first – think later”
practice reduces the number of repeated analysis of the original samples.
Q-TOF MS and Residue AnalysisJinchuan Yang, Ken Rosnack, Joseph Romano Waters Corporation, Milford, MA, USA
2Q-TOF MS and Residue Analysis
This “collect first – think later” idea is further improved by Waters’ patented MSE technique, which collects not
only the accurate mass spectra of all of the molecular ions, but also the fragment ions of the sample during the
data acquisition. MSE does this by switching between low and high collision energy in the collision cell during
the data acquisition. This allows all of the accurate masses of molecular ions and fragment ions, as well as their
chromatographic retention times, to be collected in a single run. These interrelated retention time-aligned
molecular and fragment ion accurate mass spectral data, generated only by Waters patented Q-Tof MSE technique,
provide a unique solution to resolve potential co-eluting isobaric, or even isomeric ions. Other MS instruments, no
matter how high its mass resolution is, cannot resolve co-eluting isomeric ions based on mass resolution alone.
Figure 1 illustrates the benefits of MSE for identifying simetryn and desmetryn isomers.3
Figure 1. Simetryn and desmetryn are isomeric and co-elute, but have unique MSE fragment ions (circled) that can be used to unequivocally identify them.3
Desmetryn fragment ions
Simetryn standardm/z 214.1126
Simetryn fragment ions
Desmetryn standardm/z 214.1126
EU regulation
EU Commission Decision 2002/657/EC4 and Guidance SANCO/10684/20095 are important regulations
for residue analysis in the European Union community. The document 2002/657/EC provides guidance on
performance criteria, requirements, and procedures for analytical methods concerning substances and residues
in live animals and animal products, while the SANCO/10684/2009 document provides method validation
and quality control procedures for pesticide residue analysis in food and feed. There is a partial overlap in
coverage areas for these two guidelines, since some food and feed products are of animal origin.
Decision 2002/657/EC, which was implemented in 2002, specifies performance criteria and requirements
for the screening and confirmatory analytical methods and validation procedures. According to this guideline,
the analytical screening methods should have less than 5% (b-error) false compliant rate (or false negative).
For the confirmatory methods for organic residues and contaminants, chromatographic analysis coupled to
mass spectrometer or other detectors (such as IR, fluorescence, full-scan DAD, etc.) can be used. When MS is
used, the minimum requirements for the relative ion intensities (ion ratios), full scan MS spectral data, and
non full-scan MS data are specified in the Decision 2002/657/EC. It should be pointed out that a system of
identification points was introduced for non full-scan MS data, such as SIM (selected ion monitoring) or MRM,
as shown in Table 1. High resolution MS (HRMS) earns more identification points than low resolution MS per
diagnostic ion; however, the guideline lacks the necessary mass accuracy requirement for HRMS.
3Q-TOF MS and Residue Analysis
MS technique Identification points earned per ion
Low resolution mass spectrometry (LR) 1,0
LR-MSn precursor ion 1,0
LR-MSn transition products 1,5
HRMS 2,0
HR-MSn precursor ion 2,0
HR-MSn transition products 2,5
Guide SANCO/10684/2009, which was implemented in 2010, provides guidelines for method validation
and quality control procedures, from sampling, transport, processing and storage of samples, to reporting of
results. Its criterion for screening methods is also less than 5% b-error in false negative rate, the same as
2002/657/EC. It contains more specific requirements on the MS data for the confirmation or identification
of potential positive results, described in Table 2, than the decision 2002/657/EC does. The benefits of high
resolution full scan MS data is also recognized in that it allows fewer HRMS diagnostic ions for confirmation.
In addition, the mass accuracy requirement (< 5 ppm) is specified for HRMS. No identification point system is
used, however, in SANCO/10684/2009.
MS mode
Single MS
(standard mass
resolution)
Single MS
(high resolution/high
mass accuracy
MS/MS
Typical systems
(examples)quadrupole, ion trap, time-of-flight (TOF)
TOF, Orbitrap, FTMS, magnetic sector
Triple quadrupole ion trap, hybrid MS (e.g. Q-TOF, Q-trap)
Acquisition
Full scan, limited m/z range, Selected ion monitoring (SIM)
Full scan, limited m/z range, Selected ion monitoring (SIM)
Selected/multiple reaction monitoring (SRM/MRM), full scan product-ion spectra
Requirements for
identification
≥ 3 diagnostic ions, (preferably including the quasi molecular ion)
≥ 2 diagnostic ions (preferably including the quasi molecular ion). Mass accuracy < 5 ppm. At least one fragment ion.
≥ 2 product ions
Table 1. The relationship between a range of classes of mass fragment and identification points earned in Commission Decision 2002/657/EC.
Footnotes:
1. Each ion may only be counted once.
2. GC-MS using electron impact ionization is regarded as being a different technique to GC-MS using chemical ionization.
3. Different analytes can be used to increase the number of different points only if the derivatives employ different reaction chemistries.
4. For substances in Group A of Annex 1 to Directive 96/23/EC, if one of the following techniques are used in the analytical procedure: HPLC coupled with full-scan diode array spectrophototometry (DAD); HPLC coupled with fluorescence detection; HPLC coupled to an immunogram; two-dimensional TLC coupled to spectrometric detection; a maximum of one identification point may be contributed, providing that the relevant criteria for these techniques are fulfilled.
5. Transition products include both daughter and granddaughter products.
Table 2. Identification requirements for different types of mass spectrometers in SANCO/10684/2009.5
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References
1. http://www.codexalimentarius.net/web/index_en.jsp
2. J Morphet, P Hancock. A rapid method for the screening and confirmation of over 400 pesticide residues in food. Waters Application Note, no. 720002628en, 2008.
3. E Riches, J Morphet, P Silcock, P Hancock, H Major. Rapid and sensitive targeted TOF screening for pesticides in environmental waters. Waters Application Note no. 720003671en, 2010.
4. Commission Decision 2002/657/EC. Off J EurCommun. L221, 8 (2002).
5. Method validation and quality control procedures for pesticide residues analysis in food and feed. Document no. SANCO/10684/2009: http://ec.europa.eu/food/plant/protection/pesticides/publications_en.htm
6. P. Hancock, J Morphet, E Cognard, D Ortelli. Screening veterinary drugs in products of animal origin. Waters Application Note, no. 720003189en, 2009.
7. F Hernandex, S Grimalt, O J Pozo, J V Sancho. Use of ultra-high-pressure liquid chromatography-quadrupole time-of-flight MS to discover the presence of pesticide metabolites in food samples. J Sep Sci. 32: 2245-2261, 2009.
8. A Kaufmann, P Butcher, K Maden, M Widmer. Quantitative multi-residue method for about 100 veterinary drugs in different meat matrices by sub 2-µm particulate high-performance liquid chromatography coupled to time of flight mass spectrometry. J Chromatogr A. 1194: 66-79, 2008.
9. D Ortelli, E Cognard, P Jan, P Edder. Comprehensive fast multi-residue screening of 150 veterinary drugs in milk by ultra-performance liquid chromatography coupled to time of flight mass spectrometry. J Chromatogr B. 877: 2363-2374, 2009.
High mass resolution in MS
In both regulations, the benefit of high resolution MS for confirmation is
recognized. However, the definitions of HRMS in both regulations are not
consistent. The earlier regulation (2002/657/EC) defines HRMS as the resolution
typically > 10,000 at 10% valley, which is commonly believed equivalent
to 20,000 FWHM (full width at half maximum), while the newer regulation
(SANCO/10684/2009) defines it as > 10,000 FWHM. In spite of the ambiguity
of the HRMS definition, Q-TOF MS instruments of various mass resolving power
have been successfully applied to residue analysis of veterinary drugs and
pesticides in food matrices.6-9
SUMMA RY
A tandem quadrupole mass spectrometer coupled to an LC is the most popular
technique in targeted organic residue analysis. The development of Q-TOF MS
technology, however, provides an attractive alternative MS approach in organic
residue analysis. It has good sensitivity, selectivity, improved dynamic range,
speed, and it can handle virtually an unlimited number of multi-residue analytes
in a single run. Its accurate mass spectral data can be retrospectively processed
for new emerging compounds of interest, and its non-targeted analysis capability
can not be matched by a tandem quadrupole LC-MS/MS approach.
Commission Decision 2002/657/EC and Guide SANCO/10684/2009 provide
criteria and minimum requirements for method validation and analytical quality
control procedures for residue analysis. These guidelines recognize the benefits
of high resolution and accurate mass MS data for residue analysis, although the
definitions of high resolution MS are not unified.
Besides the high mass resolution and accurate mass, it is worth pointing
out that other important factors, such as ion fragmentation, dynamic range,
sensitivity, acquisition speed, data processing software, sample extraction,
and LC separation, all play important roles in the screening and confirmation
in residue analysis. Instead of relying mainly on the high mass resolution of the
mass spectrometer, a good analyst should take all these factors into consideration
and adopt a systematic approach to achieve the best overall performance in the
residue analysis.